Project Summary/Abstract The goal of this work is to characterize a unique small molecule probe of nuclear protein TOX (thymocyte selection-associated HMG box protein) to aid in understanding how this protein regulates development and function of the immune system. These studies would also provide a significant proof-of- principle that a small molecule can target the interface of a protein and the DNA minor groove. The TOX amino acid sequence is 94% identical between mice and humans, suggesting a highly conserved function. Our laboratory has previously demonstrated that many aspects of the immune system fail to develop in TOX-deficient mice, including CD4 T lymphocytes and the entire innate lymphoid cell lineage, due to a failure of specific progenitor cells to continue differentiation in thymus and bone marrow, respectively. We focus here on the DNA binding HMG-box domain of TOX and its role in regulating gene expression. This is a highly evolutionarily conserved region of the protein, and one that is shared with the other three members of the TOX subfamily of proteins. Thus, the small molecule probe we have identified has potential to have broad utility in a number of biological contexts. We have determined the crystal structure of the HMG-box domain of TOX and used these data and molecular docking to identify a small molecule, neurodazine, that we predicted would bind TOX and alter the interaction of the protein with DNA. Neurodazine is an imidazole-based cell-permeable small molecule that was identified by its ability to alter gene expression associated with neuronal cells, but has not been well studied. Here we will address whether this small molecule is active as a modulator of TOX activity. That neurodazine can influence TOX activity was suggested by novel assays we developed to detect TOX binding to DNA, and to allow an easy read out of TOX-mediated regulation of gene expression. We propose here to characterize the binding of neurodazine to TOX, including crystal structure determination of the complex, and to determine whether neurodazine specifically inhibits TOX-induced gene regulation. Most interestingly, neurodazine can enhance binding of TOX to DNA, but appears to disrupt the function of the protein. This has led to the hypothesis that neurodazine may stabilize TOX on chromatin, altering its nuclear dynamics. This will be tested in living cells using fluorescence recovery after photobleaching. Finally, two distinct cell systems will be used to determine the potential of neurodazine to inhibit TOX activity in the context of immune cells; bone marrow progenitor cell differentiation to innate lymphoid cells and TOX- dependent cutaneous T cell lymphoma cell growth. Together, these studies will provide key insights into structure-function relationships of this protein and form the basis for future development of additional small molecule modulators of the TOX-family of proteins that could be useful as probes and leads for pretherapeutics.